Eukaryotic cells have evolved a complex set of regulatory proteins to maintain genome stability by regulating DNA replication once and once per cell cycle. Despite intensive investigation, a precise understanding of the proteins that are phosphorylated during the initiation phase of DNA replication remains to be identified. Here we investigate the roles of cyclin E and cyclin A in the G1 to S-phase transition and reconstitute initiation of DNA replication in cyclin depleted nuclei using an in vitro DNA replication system.
Using a murine 3T3 fibroblast model, chemical and genetic inhibition of CDK activity was used to evaluate the role of specific cyclin complexes in post-quiescent cells. CDK inhibition reduced levels of CDC6, MCM2 and PCNA in all contexts. Evaluation of the role of individual cyclin-CDK complexes was performed by siRNA mediated depletion. Cyclin E1/E2 depletion showed that Cyclin E regulates CDC6, MCM2 and PCNA at the transcriptional level; whereas Cyclin A regulates their accumulation though protection from the ubiquitin -proteasome system in late G1 phase. Furthermore, ubiquitination of CDC6 and MCM2 was confirmed using Histagged ubiquitin pulldown assays suggesting that the CDK and the UPS regulate their levels late G1 phase.
Comparison of replication competence was performed using in vitro cell free DNA replication assays. Cyclin E depleted nuclei were unable to initiate DNA replication in S phase extracts; whereas cyclin A2 depleted nuclei retained full activity. Crucially, cyclin E depleted nuclei were unable to load CDK/DDK activated MCM2 proteins onto chromatin in G1 phase that correlated with replication competence in vitro. The data suggests that sustained cyclin activity is required to maintain pre-RC protein levels and facilitate formation of replication complexes in G1 phase. These results further suggest that perturbations in the CDK network results in destabilisation of replication proteins CDC6, MCM2, Ciz1 and PCNA that blocks replication complex assembly on chromatin, contributing to genomic stability.